Fig. 6. SLC25A22 drives glutamine-dependent CXCL1 via asparagine.

a In glutamine-depleted (12 h) DLD1 cells, addition of glutamine (72 h) dose-dependently induced CXCL1 mRNA. SLC25A22 knockout selectively inhibited CXCL1 mRNA at high glutamine levels (n = 3). Each dot represents an independent sample. b After glutamine depletion (12 h), glutamine (72 h) dose-dependently induced CXCL1 secretion in DLD1 cells and was impaired by SLC25A22 knockout (n = 3) (left). In glutamine-depleted (12 h) CT26 cells, glutamine (24 h) dose-dependently induced Cxcl1 secretion. SLC25A22 knockout inhibited Cxcl1 secretion at high glutamine (n = 3) (right). Each dot represents an independent sample. c Transaminase inhibition by aminooxyacetate (AOA, 100 μM) inhibited CXCL1 mRNA; whereas blockade of glutamate dehydrogenase 1 (GDH1) by Purpurin (25 μM) and R162 (25 μM) had an opposite effect (n = 3). A similar effect was observed for CXCL1 secretion (n = 3). Each dot represents an independent sample. d Schematic diagram showing the metabolic outputs of glutamine via SLC25A22. e Supplementation of downstream metabolites (2 mM, 24 h) (α-KG, dimethyl α-ketoglutarate; Succ, dimethyl succinate; OAA, dimethyl oxaloacetate; Asp, aspartate; Asn, asparagine) showed that asparagine restored CXCL1 mRNA in SLC25A22 knockout DLD1 cells (n = 3). Aspartate restored CXCL1 mRNA expression in DLD1-SLC-KO cells overexpressing SLC1A3 (n = 3). Each dot represents an independent sample. f Asparagine (2 mM, 24 h) restored CXCL1 secretion in both DLD1 and CT26 cells with knockout of SLC25A22 (n = 3). Aspartate restored CXCL1 secretion in DLD1-SLC-KO cells overexpressing SLC1A3 (n = 3). Each dot represents an independent sample. g After depleting glutamine (12 h), [¹³C₅]-Glutamine stable isotope labeling and LC-MS was performed in DLD1 and CT26 cells with or without SLC25A22 knockout. Total and ¹³C-labeled asparagine were reduced by SLC25A22 knockout (n = 3). Each dot represents an independent sample. h Treatment with asparagine (2 mM, 24 h) restored the capacity of DLD1-SLC-KO and CT26-Slc-KO cell conditioned medium to promote MDSC migration, without direct effects on MDSC migration (n = 4). i Validation of ASNS knockdown by western blot (left). LC-MS of DLD1 and CT26 cells co-transfected with siASNS and SLC1A3, followed by incubation with ¹³C₄-aspartate (2 mM, 96 h) confirmed the ASNS blockade (n = 4). Each dot represents an independent sample. j LC-MS showed that Asparaginase (ASNase) (24 h) depleted cellular asparagine in DLD1 and CT16 cells (n = 4). Each dot represents an independent sample. k siASNS (upper) or ASNase (lower) reduced CXCL1 mRNA and l secretion in DLD1 and CT26 cells (n = 3). Each dot represents an independent sample. m Conditioned medium from siASNS (upper) or ASNase-treated (lower) DLD1 and CT26 cells had reduced ability to induce MDSC migration. (n = 4). Each dot represents an independent sample. Data are shown as mean ± SD (a–c, e–m) and ± SEM for metabolite curves g. Two-tailed one-way ANOVA (a–c, e, g–m). Two-tailed Student’s t test f. ns, no significance. Source data are provided as a Source Data file.